Century-Old DNA Barcodes Reveal Phylogenetic Placement of the Extinct Jamaican Sunset Moth, Urania Sloanus Cramer (Lepidoptera: Uraniidae)
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RESEARCH ARTICLE Century-Old DNA Barcodes Reveal Phylogenetic Placement of the Extinct Jamaican Sunset Moth, Urania sloanus Cramer (Lepidoptera: Uraniidae) Vazrick Nazari1*, B. Christian Schmidt1, Sean Prosser2, Paul D. N. Hebert2 1 Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa Research and Development a11111 Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada, 2 Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada * [email protected] Abstract OPEN ACCESS Analysis of the DNA barcode region of the cytochrome c oxidase 1 gene from a specimen Citation: Nazari V, Schmidt BC, Prosser S, Hebert of the extinct Jamaican sunset moth, Urania sloanus, places this species as a sister to the PDN (2016) Century-Old DNA Barcodes Reveal Central American U. fulgens. We found that all Urania F. species were closely related Phylogenetic Placement of the Extinct Jamaican Sunset Moth, Urania sloanus Cramer (Lepidoptera: (<2.8% maximum divergence at COI), with the Cuban endemic U. boisduvalii appearing as Uraniidae). PLoS ONE 11(10): e0164405. sister to the rest. The low divergence in DNA barcodes and genitalic structures indicate that doi:10.1371/journal.pone.0164405 the Cuban U. poeyi and eastern Brazilian U. brasiliensis are geographic segregates of U. Editor: Dorothee Huchon, Tel Aviv University, fulgens and U. leilus respectively, so the former two taxa are accordingly recognized as ISRAEL subspecies. Received: June 7, 2016 Accepted: September 23, 2016 Published: October 20, 2016 Copyright: © 2016 Nazari et al. This is an open access article distributed under the terms of the Introduction Creative Commons Attribution License, which permits unrestricted use, distribution, and The Jamaican Sunset moth, Urania sloanus (Cramer, 1776), often regarded as one of the most reproduction in any medium, provided the original beautiful of all moths, became extinct just 116 years after its description [1,2]. Endemic to author and source are credited. Jamaica, it inhabited low-elevation tropical rainforest containing its host plant Omphalea L. Data Availability Statement: All new sequences (Euphorbiaceae), especially O. triandra L. (Jamaican Cobnut) and O. diandra L. [3–5]. Urania are now deposited on GenBank (accession sloanus was named by Dutch entomologist Pieter Cramer [6] in honor of Sir Hans Sloane numbers KX781954-KX781991). Additional (1660–1753), an English naturalist and collector who explored Jamaica from 1687 to 1689, sequences used in Supporting Info S1. DOI for the recording and illustrating the species for the first time in his travel diaries published in 1725 dataset: dx.doi.org/10.5883/DS-URANIA. [7]. The life history and behaviour of U. sloanus was subsequently described in detail by Philip Funding: This study was supported by grants to Gosse [3,8–9], who reported that adults were common from mid-March to late July but usually Paul D.N. Hebert (PDNH) from Natural Sciences rare in the winter. Periodic episodes of abundance, when swarms of these moths occurred at and Engineering Research Council of Canada flowering trees in the Blue Mountains of Portland, were intervened by years of great scarcity (NSERC) and from the Government of Canada through Genome Canada and the Ontario [1,8,10–11]. The moth became increasingly rare in the early 1890s; the last sighting occurred in Genomics Institute in support of the International 1894–1895 [1], although the species may have survived until 1908 [12]. Lees and Smith [11] Barcode of Life Project. postulated that populations of U. sloanus declined below a sustainable level due to the loss of PLOS ONE | DOI:10.1371/journal.pone.0164405 October 20, 2016 1 / 13 Phylogeny of Urania (Lepidoptera: Uraniidae) Competing Interests: The authors have declared its main larval host through habitat destruction linked to agriculture or hurricanes. Vinciguerra that no competing interests exist. [12] suggested the species may still persist in remote areas with appropriate habitat and host plants. In addition to U. sloanus, five other species of Urania Fabricius are generally recognized: U. leilus (Linnaeus, 1758), widespread in tropical South America east of the Andes; U. fulgens Walker, 1854, a migratory species distributed from Mexico through Central America to north- western South America (occasionally straying to Florida and Texas [13] as well as Jamaica [Lees, pers. comm.]), and U. boisduvalii Guérin-Meneville, 1829, endemic to Cuba. The status of two other species, namely U. brasiliensis (Swainson, 1833) (Atlantic coast of Brazil) and U. poeyi (Herrich-Schäffer, 1866) (Cuba) is controversial as they have been treated either as sepa- rate species [11,14–17], or subspecies or synonyms of U. fulgens and U. leilus, respectively [18]. Urania fulgens can exhibit forms that are externally very similar to U. leilus, and these two taxa have also been suggested to be conspecific [19]. By contrast, only U. sloanus shows pink and red coloration on the dorsal hindwing. The diagnostic value of genitalic structures has not been evaluated in Urania. In addition to the Neotropical genus Urania, the pantropical subfamily Uraniinae contains six other genera (Alcides Hübner, Chrysiridia Hübner, Cyphura Warren, Lyssa Hübner, Urap- teritra Viette and Urapteroides Moore) with many large, tailed diurnal species with a spectacu- lar phenotype. The family Uraniidae apparently originated in the late Cretaceous, ~75 Mya [20] from a common ancestor with the Sematuridae [21–22] although no comprehensive phy- logeny is available for it. Lees and Smith [11] provided a preliminary phylogeny for the Uranii- nae based on seven larval and adult tympanic characters, while molecular studies [20–22] have incorporated a few representative uraniid genera within broader lepidopteran phylogenies. Urania and Chrysiridia were resolved as sister genera by Lees and Smith [11] based on shared larval and tympanic morphology and further supported by the diurnality of their adults, wing scale iridescence, and their shared use of Omphalea as larval hosts. A combined molecular and morphological analysis [22], however, found weak support for Alcides as the sister group to Urania, with both sisters to Chrysiridia. Urania moths are unique among Lepidoptera in having stridulatory organs on their male prothoracic legs [23–25]. They are also among the very few moths that form migrant popula- tions, being chemically defended against predators by polyhydroxy alkaloids sequestered from their host plants, Omphalea [26–27]. Species in the same genus are also hosts for Afrotropical Chrysiridia, as well as the Indo-Australian Alcides and Lyssa [11,28]. In fact, the disjunct distri- butions of these moths almost exactly correspond to the global range of Omphalea, suggesting a deep historical relationship, perhaps even predating the final break-up of Gondwanaland in the Cretaceous, ca. 100–65 Mya [11]. All Urania species are allo- or parapatric with respect to each other and show island ende- mism (U. boisduvalii, U. poeyi, U. sloanus), but species relationships have never been studied. Here we investigate the pattern of sequence divergence at COI among species of Urania, including the extinct U. sloanus, in the large context of family Uraniidae. Methods and Materials Samples 40 specimens from all six recognized species of Urania, including one specimen of U. sloanus, were sequenced for COI. In addition, to aid phylogenetic inferences we included 14 representa- tives from the other six genera of Uraniinae (7), subfamilies Auzeinae (2), Epipleminae (2) and Microniinae (2), as well as family Sematuridae (1), recognized as sister to Uraniidae [21]. Sequences were downloaded from GenBank or BOLD with permission (S1 Table). Genitalia PLOS ONE | DOI:10.1371/journal.pone.0164405 October 20, 2016 2 / 13 Phylogeny of Urania (Lepidoptera: Uraniidae) were prepared following the methods of Lafontaine [29]. Male genitalia of all Urania taxa except U. sloanus were examined (n = 5); females were not available for all species. Cleaned, stained genitalia were stored and examined in 30% ethanol, and slide-mounted in Euparal before being photographed using a Leica DFC450 digital camera and the Leica Application Suite 4.7.1 software package. Amplification and sequence analysis A single leg was removed from each specimen and sent to Biodiversity Institute of Ontario in Guelph, Canada for DNA extraction, amplification, and sequencing. Non-destructive DNA extraction was used for the leg from U. sloanus. All work was performed in a dedicated clean lab using UV sterilized equipment to minimize the risk of contamination. DNA extraction and purification was performed following the methods of Prosser et al [30] and the 658 bp COI bar- code region was amplified for both Sanger-based sequencing and next-generation sequencing (NGS). For Sanger sequencing, non-tailed primers (S2 Table) were used to amplify six short, overlapping fragments using a modified version of the method described in Lees et al [31]. Each of the six fragments from each sample were bi-directionally sequenced on an ABI 3730XL capillary sequencer (Applied Biosystems) and the resulting traces were edited and assembled into full contigs using CodonCodev. 3.0.1 (CodonCodeCorporation). Because the failure of any of the six fragments to amplify or sequence resulted in missing barcode data (S3 Table), we also sequenced each sample via NGS using the method described in Prosser et al [30]. Briefly, instead of amplifying six overlapping amplicons